Interaction between â-Lactoglobulin and Phospholipids atthe Air/Water Interface

Martin A. Bos†

Food Technology, University of Lund, P.O. Box 124, S-221 00 Lund, Sweden

Tommy Nylander*

Physical Chemistry 1 and Food Technology, University of Lund, P.O. Box 124,S-221 00 Lund, Sweden

Received July 31, 1995. In Final Form: February 9, 1996X

In the present studywe used the film balance technique to study the interaction between â-lactoglobulinand distearoylphosphatidic acid, distearoylphosphatidylcholine, and dipalmitoylphosphatidic acid at theair/water interface. ThepHand the salt concentrationof the subphasewere chosenasvariables todeterminethenature of the interaction. Thearea increaseuponadsorptionat constant surfacepressurewasmeasuredas a function of the surface pressure and analyzed by applying a simple first-order kinetic model. Thehighest rate of adsorption was found for adsorption into distearoylphosphatidic acid monolayers. Theresults showthatnot only electrostatic but alsohydrophobic interactionsare important for the incorporationof â-lactoglobulin into phospholipid monolayers. The results were found to agree qualitatively with ourprevious study on the interaction of â-lactoglobulin with the same phospholipids dispersed in aqueoussolution.

IntroductionProteins and phospholipids are widely used as emulsi-

fiers or stabilizers in emulsions or foams, especially in thefood industry. Due to their easy access, their functionalproperties and nutritional value, whey proteins andphospholipids are commonly used.1,2 The major wheyprotein,â-lactoglobulin,whichhasbeenwell characterizedchemically and physically,3-8 makes a significant contri-bution to the emulsification and foaming of a variety ofdairy-based processed foods like ice cream and milkshakes. Phospholipids stabilize emulsions by their am-phiphilic character and their high capability to lower theinterfacial tension. Ithasbeenshownthatâ-lactoglobulinis able to bind phospholipids, fatty acids, triglycerides,and retinol.9-12 This binding ability has been related toa possible physical function of â-lactoglobulin; it acts asa carrier of retinol and fatty acids.10,13 From this point of

view, Kurihari and Katsuragi14 results showing that alipid protein complex between â-lactoglobulin and phos-phatidic acid inhibits bitter taste could be explained. Itis, however, not clear whether â-lactoglobulin transportsthe lipid that binds to the taste sensor or the lipid-proteincomplex itself binds to the taste sensor. This phenomenonmight be related to the observation that the complexformed by â-lactoglobulin and surfactants could beresponsible for the destabilization of emulsions.15

In thepresent study,weused the filmbalance techniqueto study the incorporation of â-lactoglobulin into variousmonolayers of syntheticallymadeandverypuredistearoylphospholipids and dipalmitoylphoshatidic acid in orderto get a better understanding of the interaction betweenâ-lactoglobulin and phospholipids at the liquid interface.The area increase, due to adsorption of â-lactoglobulin,was recorded at constant surface pressures as a functionof pH and salt concentration. We will use a simple first-orderkineticmodel,which takes only the surfacepressurebarrier into account, to compare the interfacial behavior.In the Discussion special attention will be given to theeffect of the headgroup and the hydrocarbon chain lengthaswell as the structure of the lipid-protein film. Finally,the data will be compared with dispersed phospholipidsystems as reported by Kristensen et al.16

Materials and Methods

Materials. All water used was ion exchanged, distilled, andfurther purified by aMilliQwater purification system (MilliporeCorporation,Bedford,MA). Thepurifiedwater showednobubblepersistence, and the resistance was >18 MΩ/cm.

â-Lactoglobulin (L-0130, lot91H-7005), three timescrystallizedand lyophilized, was obtained from Sigma (St. Louis, MO) andused without further purification.Syntheticallymadedistearoylphosphatidylcholine (DSPC) (lot

B9537),distearoylphosphatidylethanolamine (DSPE) (lotB9705),distearoylphosphatidylglycerol (DSPG) (lot B13142/2), distear-

* Corresponding author.† Present address: Department of Food Science, Wageningen

AgriculturalUniversity, P.O.Box 8129, 6700EVWageningen, TheNetherlands.

X Abstract published in Advance ACS Abstracts, May 1, 1996.(1) Hambling, S. G.; McAlpine, A. S.; Sawyer, L. In Advanced Dairy

Chemistry. Vol. 1: Proteins; Fox, P., Ed.; Elsevier Applied SciencePublishers Ltd.: London, 1992; pp 141-190.

(2) Walstra, P.; Jenness, R.Dairy chemistry and physics; JohnWiley& Sons: New York, 1984.

(3) Braunitzer, G.; Chen, R.; Schrank, B.; Stangl, A. Hoppe-Seyler’sZ. Physiol. Chem. 1972, 353, 832.

(4) Braunitzer, G.; Chen, R.; Schrank, B.; Stangl, A. Hoppe-Seyler’sZ. Physiol. Chem. 1973, 354, 867-878.

(5) Nozaki, Y.; Bunville, L. G.; Tanford, C. J. Am. Chem. Soc. 1959,81, 5523-5529.

(6) Timasheff, S. N.; Townend, R. Nature 1964, 203, 517-519.(7) Green, D. W.; Aschaffenburg, R.; Camerman, A.; Coppola, J. C.;

Dunnill, P.; Simmons, R. M.; Komorowski, E. S.; Sawyer, L.; Turner,E. M.; Woods, K. F. J. Mol. Biol. 1979, 40, 375-397.

(8) Townend, R.; Kumosinski, T. F.; Timasheff, S. N. J. Biol. Chem.1967, 242, 4538-4545.

(9) Diaz de Villegas, M. C.; Oria, R.; Salva, F. J.; Calvo, M.Milchwissenschaft 1987, 42, 357-358.

(10) Papiz,M.J.;Sawyer,L.;Eliopoulos,E.E.;North,A.C.T.;Findlay,B. C.; Sivaprasadarao, R.; Jones, T. A.; Kraulis, P. J.Nature 1986, 324,383-385.

(11) Puyol, P.; Perez,M. P.; Peiro, J.M.; Calvo,M.J.Dairy Sci. 1994,77, 1494-1502.

(12) Fugate, R. D.; Song, P.-S. Biochim. Biophys. Acta 1980, 625,28-42.

(13) Sawyer, L. Nature 1987, 327, 659.(14) Kurihara, K.; Katsuragi, Y. Nature 1993, 365, 213-214.(15) Tornberg, E.; Lundh, G. J. Colloid Interface Sci. 1981, 79, 76-

84.(16) Kristensen, A.; Nylander, T.; Paulsson, M.; Carlsson, A. Int.

Dairy J., in press.

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S0743-7463(95)00640-8 CCC: $12.00 © 1996 American Chemical Society

oylphosphatidic acid (DSPA) (lot B11144), dipalmitoylphospha-tidic acid (DPPA) (lot B9475), and dimyristoylphosphatidic acid(DMPA) (lot B9538) of 99+% purity were kindly provided byGenzyme Pharmaceutical and Fine Chemicals, Haverhill, U.K.All other chemicals usedwereMerckproducts of analytical gradeand were used without further purification.Methods. A KSV 5000 LB film balance system (KSV

Chemicals, Helsinki, Finland) was used to study the interfacialbehavior of the different phospholipids and â-lactoglobulin andthe interactionbetween them. Thesurface tensionwasmeasuredaccording to theWilhelmy plate method, using a platinum plateof 2 cm width. Prior to the measurement the plate was cleanedby glowing it twice in a flame.Surface pressure-area (Π-A) isotherms were recorded as

follows. Initially, distilled water or buffer solution was takeninto the trough (maximum area 150 × 675 mm2). The surfacewas cleaned by sweeping it with a Teflon barrier. Any surfaceactive components or contaminants were removed by suctionfrom the interface. To check the cleanness, a Π-A isotherm ofthe bare interface was recorded under the same conditions usedwhen recording the isothermsof thephospholipids. The cleaningprocess was repeated until the measured surface pressure wasbelow 0.1 mN/m upon complete compression of the film. Justbefore starting the experiment, the Teflon barrier was moved tothemaximumsurface area. Thephospholipidswere spread froma spreading solvent consisting of chloroform/methanol (2:1 v/v%). In the case of phosphatidic acid a few drops of concentratedHCl was also added. The phospholipid solution, 250 µL contain-ing 0.5 mg/mL, was carefully applied at the interface by amicrosyringe of which the tip was placed at a low angle to theinterface in such a way that a large meniscus was formed underthe needle. To avoid solvent effects on the structure, â-lacto-globulin was spread from crystals, giving a total amount of 30-40 µg. A time span of 5 min was allowed for evaporation of thesolvent and to ensure complete spreading of the crystals, beforethe compression at a speed of 5 cm/min was started.In the adsorption experiments (Π versus time dependencies)

the â-lactoglobulin solution was put into the trough (depth 0.6cm). The surface was cleaned by sweeping the Teflon barrierand by suction of the interface. The Wilhelmy plate was thenimmediately lowered into the solution, and the adsorption ofâ-lactoglobulin versus timewas followedby recording the surfacetension.The adsorption of â-lactoglobulin in the presence of a phos-

pholipid monolayer was performed by applying the followingmethod. The lipids were spread as described before, directlyonto the cleaned interface of the protein solution. The layer wasthen immediately compressed to a high surface pressure of 35-40 mN/m, to squeeze out the proteins from the lipid monolayer.Afterkeeping thishighpressure for 5min, theareawas increaseduntil the surfacepressure to study the incorporationof theproteininto the phospholipid monolayer was reached. Keeping thesurface pressure constant, the area increase, as a result of theincorporation of â-lactoglobulin in the phospholipid monolayer,was recorded versus time for 25-30 min. This procedure wasthen repeated for the next surface pressure to be studied.All experimentswere performed at a temperature of 25 ((0.2)

°C using a thermostat, and deviations between duplicate experi-ments were e0.4 mN/m for Π versus time measurements ande0.3 mN/m for the Π versus area isotherms.

Results

Before the penetration of â-lactoglobulin into phospho-lipid monolayers was investigated, the effects of thesolution conditions used aswell as the protein adsorptionat the air/aqueous interface were thoroughly studied.

Π-A Isotherms of Phospholipids. The effect of thesubphase composition on the Π-A isotherms for thevarious phospholipids are shown in Figures 1 and 2. Thespreading of the phospholipids was quantitative underthe used experimental conditions, as different amountsspreadgave the sameΠ-A isotherm. The isothermswererecorded at a compression speed of 5 cm/min, although noeffect of compression speed in the range 2-10 cm/minwas found. Extrapolationof thesteeppartof the isotherms

toΠ ) 0 gives the so-called limiting area per phospholipidmolecule. These areas obtained at 25 °C for variousphospholipids are summarized in Table 1. The limitingarea seems to reflect the size of theheadgroup of the phos-pholipids: DSPC>DSPE≈DSPG>DSPA≈DPPAwhendistilled water was the subphase. These areas changedsomewhat when buffers were used, but DSPC still gavethe largest area per molecule. For the two zwitterionicphospholipids DSPC and DSPE the headgroup area wasindependent of thepHof the subphaseused. Thenegativephosphatidic acid gave aheadgroupareawhichwas loweron water than on a subphase with higher ionic strength.It should be noted that the spreading of DPPA and DSPAwas made from solutions where the lipid was uncharged(HCl was added to the spreading solvent). Thus in theabsence of buffer a lower degree of dissociation of thephosphatidic acid headgroup is likely, and consequentlyless repulsion exists between the headgroups, giving asmaller area per molecule (Table 1). The reverse wasobserved for DSPG, where the largest area is obtainedwhen the lipid was spread on pure water.

Π-A Isothermsof â-Lactoglobulin. Figure 3 showstheΠ-A isotherms forâ-lactoglobulinwithdistilledwaterand 10mMphosphate buffer pH 4 and pH 7 as subphase,respectively. The proteinwas spread from small crystalsonto the surface. Extrapolation of the steep part of theisotherm before Π ) 18 mN/m to Π ) 0 gave the limitingarea for the molecules: 29, 28, and 30 nm2 for water, pH4.0, and pH 7.0, respectively. These values correspond toan adsorbed amount of about 1 mg/m2, which indicates

Figure 1. Surface pressure (Π) as a function of the moleculararea for different distearoyl phospholipid monolayers: (a)Subphase, distilled water; (b) subphase, 10 mM phosphatebuffer, pH 4; (c) subphase, 10 mM phosphate buffer, pH 7.

2792 Langmuir, Vol. 12, No. 11, 1996 Bos and Nylander

efficient spreading.17 It is noteworthy that this area isabove twice the one (≈12 nm2) expected for a close packedlayer of monomers as estimated from the moleculardimensions.1,2 This indicates that the protein adopts adifferent (unfolded) conformation at the air/aqueousinterface compared to in crystals or in solution. At about18 mN/m a discontinuity in the isotherms is observed.Above this surface pressure whole protein molecules orsegments of the protein molecules are probably squeezeddown into the subphase.18-20

Adsorptionofâ-Lactoglobulinat theAir/AqueousInterface. A protein concentration which is low enough

to allow for the spreading of the phospholipids when nosurface pressure barrier from the adsorbed proteinmolecules is present had to be found. Therefore, theadsorption of â-lactoglobulin versus time for differentprotein concentrations was investigated (Figure 4). Theresults showed that for aprotein concentration of 1.2mg/Lthe surface pressure started to increase after 5min. Thislag time is enough to spread the phospholipids at the air/water interface and compress the lipid layer to thedesiredsurfacepressurebeforean increase of the surfacepressuredue to theadsorptionofproteinmoleculesoccurs. It shouldbe borne in mind that even though no surface pressure isobserved a substantial amount of protein can be presentat the interface, as illustrated by the Π-A isotherm forâ-lactoglobulin (Figure3). At lowerprotein concentrationsthe adsorption kinetics was found to be too slow. Thesurface pressure reachedafter 2h of adsorption (15.5mN/m) and also the observed kinetics are in good agreementwith the results reported earlier.19,21,22 Furthermore, theΠ versus time curve shows that the plateau value of Πapproaches 20 mN/m as the protein concentration in-creases. This value is close to the point of discontinuityof the Π-A isotherm of the protein.

Π-A Isotherms of Mixed Protein-PhospholipidMonolayers. The Π-A isotherm of a spread monolayerofDSPAonasolutionofâ-lactoglobulinwill largelydependon the time elapsed between spreading and compression,as illustrated in Figure 5. Already 1min after spreading,the isothermwasshifted toward largerareaspermolecule,obviously due to the adsorption of â-lactoglobulin. Thus,as expected the increase in Π started at a larger area per(17) MacRitchie, F. Chemistry at Interfaces; Academic Press, Inc.:

London, 1990.(18) MacRitchie, F.; Alexander, A. E. J. Colloid Sci. 1963, 18, 453-

457.(19) MacRitchie, F. Colloids Surf. 1989, 41, 25-34.(20) Archer, R. J.; La Mer, V. K. J. Phys. Chem. 1955, 59, 200-208.

(21) Tornberg, E. J. Colloid Interface Sci. 1978, 64, 391-402.(22) Paulsson, M.; Dejmek, P. J. Colloid Interface Sci. 1992, 150,

394-403.

Figure 2. Surface pressure (Π) as a function of the moleculararea for different diacylphosphatidic acid monolayers: (a)subphase, distilled water; (b) subphase, 10 mM phosphatebuffer, pH 4; (c) subphase, 10 mM phosphate buffer, pH 7.

Table 1. Limiting Headgroup Area (in Å2 with aStandard Deviation of 0.3 Å2) at the Air/LiquidInterface for Various Phospholipids at 25 °Ca

DMPAb

(-)DPPA(-)

DSPA(-)

DSPC(+/-)

DSPE(+/-)

DSPG(-)

water 39 38 37 55 45 45pH 4.0 50 42 43 55 46 42pH 7.0 41 45 53 46 46

a 10 mM phosphate buffers were used unless stated otherwise.b In parentheses are the charges at pH 4.0 and 7.0 estimated fromthe data given by Marsh,37 where +/- denotes zwitterion.

Figure 3. Surface pressure (Π) as a function of the moleculararea for â-lactoglobulin on 10 mM phosphate buffer pH 4 or 7or distilled water as subphase.

Figure4. Surface pressure (Π) of â-lactoglobulin as a functionof time for various protein concentrations in 10mMphosphatebuffer pH 7 as subphase.

Interaction between â-Lactoglobulin and Phospholipids Langmuir, Vol. 12, No. 11, 1996 2793

molecule when a longer time was allowed for the adsorp-tion of â-lactoglobulin. However, the inflection point onthe isotherm, which was also present in the isotherm ofthe pure â-lactoglobulinmonolayer, occurred at the samepressure (23 mN/m), independent of the allowed time foradsorption. As the film was further compressed, theisotherms of the mixed monolayer asymptotically ap-proached that of the phospholipid monolayer. At a highenough pressure the isotherms coincided, indicating thatthe protein was completely squeezed out from the lipidmonolayer.Penetration/Adsorption of â-Lactoglobulin into

aPhospholipidMonolayer. Typical experiments of theeffect of incorporation of â-lactoglobulin into a DSPCmonolayer are shown in Figure 6. As expected the slopeof the area increase versus time curve, that is the rate ofâ-lactoglobulin penetration, decreased with time andreacheda steady state. The samebarrier position as afterthe first compression tohigh surfacepressure (35-40mN/m) was obtained after each measuring cycle when the

layer was compressed to 35-40 mN/m (see Figure 6a).This indicates that no desorption of phospholipids orphospholipid-protein complexes into the subphase oc-curred. The measured adsorption rates were also inde-pendent of the different sequences of applied surfacepressures, and the deviation between the experimentswas less than 5%.When the phospholipid was spread onto the cleaned

interface of the protein solution as described before,followed by compression of the mixed phospholipid-protein layer directly to the surface pressure, where thepenetration was studied, the same adsorption rates wereobtained. Thuswe are quite confident that the procedureof reusing the phospholipid film after compression to ahigh surface pressure is a well founded method for thesystems studied.Wehaveanalyzed our datausing twoprocedures. First

the “penetration capacity” of â-lactoglobulin, that is thesurface pressure at which the protein is unable topenetrate into the phospholipid monolayer,23 was deter-mined. Second, we will present an attempt to evaluatethe kinetics of the penetration of â-lactoglobulin from therate of area increase.Penetration Capacities. A closer look at Figure 6

reveals that the barrier position (which is proportional tothe area) versus time curve has two regions where thesteep part is simply the area increase needed to establishthe desired surface pressure. The derivatives of the areaversus time curveswere determined, and the values after25minwere chosenas the steadystatevalues todeterminethe penetration capacities. The steady state value of thearea increase perminute, dA/dt, as a function ofΠ for theadsorption of â-lactoglobulin into amonolayer of DSPC isgiven for different ionic strengths at pH7 inFigure 7. Thedatawere fitted to a linear function and the abscissa, thatis when the rate of the area increase is zero, gives thepenetrationcapacity. InTable2 thepenetrationcapacitiesof â-lactoglobulin for different phospholipid monolayersand salt concentrations at pH 4 and 7 are given.Kinetics of Penetration/Adsorption. In analogy

with a protein molecule adsorbing in its own monolayerat the air/water interface, a protein molecule adsorbingin a phospholipid monolayer will experience a surfaceenergy barrier.17 As a result not every molecule arrivingat the interface will be adsorbed. The adsorption at theinterfacewill be controlled by energy barriers (apart fromdiffusion), like the number of available sites, the initial

(23) Quinn, P. J.; Dawson, M. C. Biochem. J. 1969, 113, 791-803.

Figure 5. Surface pressure (Π) as a function of the moleculararea per lipid for DSPA and mixed â-lactoglobulin/DSPAmonolayers recorded at different times elapsed betweenspreading and compression. The protein concentration in thesubphase, 10 mM phosphate buffer, pH 7, was 1.15 mg/L.

Figure 6. Example of the adsorption of â-lactoglobulin into aDSPCmonolayeratvarioussurfacepressures (Π).Thesubphasewas a 10mMphosphate buffer, pH7, containing 150mMNaCl,and the protein concentration was 1.15 mg/L: (a) barrierpositionasa functionof time; (b) accompanyingsurfacepressure(Π) as a function of time.

Figure 7. Steady state rate of area increase as a function ofthe surface pressure (Π) for the adsorption of â-lactoglobulininto a DSPC monolayer at different amounts of added NaCl:(O) 0mM; (0) 50mM; (4) 150mM.Extrapolation toΠ ) 0 givesthe so-called penetration capacity (see text).

2794 Langmuir, Vol. 12, No. 11, 1996 Bos and Nylander

surface pressure barrier, and the electrical potentialbarrier.17 In our study, we will use a simple first-orderkinetic model, which takes only the surface pressurebarrier into account. This approach will be used as a toolto compare thekinetics of incorporation of â-lactoglobulinin various phospholipid monolayers.In order for aproteinmolecule to enter thephospholipid

monolayer, an area A0 of the interface must be cleared.For this process∫ΠdA0 has to be evaluated. If the surfacepressure is kept constant and it is assumed that there areno interactionsbetween themolecules, the integral equalsΠA0. For a first-order reaction process, the rate of theprocess (dΓ/dt) is given by a Boltzmann distribution:

where Γ is the protein surface concentration, kads is therate constant of protein adsorption, and c0 is the proteinconcentration in the bulk. If A0 is constant, eq 1 predictsa linear relationshipbetween ln(dΓ/dt) andΠ,witha slopeproportional to A0.Rates of adsorption at constant surface pressure may

be measured by the increase of area (A) as a function oftime or at constant area by the increase of the surfacepressure.17 If the rate remains constant, the logarithmof thearea increases linearlywith timeat constant surfacepressure anddΓ/dt) [lnA/t]Γ. The surface concentrationΓ can be obtained from the Π-A isotherm of the spreadmonolayer of the protein (Γ ) 1/Am), in which Am is thearea per molecule at a particular surface pressure. Itshould be pointed out that in this study the rates ofadsorption were obtained from the linear regions of theln A versus time curve, that is after 10 min of adsorption.In Figures 8 and 9 the rates of adsorption of â-lacto-

globulin into phospholipid monolayers of DSPA, DSPC,andDPPAat pH7 and 4 and different salt concentrationsare presented as ln(rate) versusΠ. A linear fit of the dataaccording to eq 1 gave correlation coefficients r g 0.98.The area A0 to be cleared for adsorption was calculatedfrom the slope and found to vary between 22 and 40 Å2.This area is about one third of the area needed foradsorption of â-lactoglobulin into its own protein layer(103 Å2)19 and much less than the area of the â-lactoglo-bulinmolecule,which is on the order of 1200Å2. Probably,adsorption of a few segments is already sufficient to gainenough free energy for adsorption. This has also beenobserved for the adsorption of several other proteins intotheir own protein monolayer.21,24,25

DiscussionEffect of thePhospholipidHeadgroup. Since very

pure andwell defined phospholipids, with the same chain

length were used in this study, differences in penetrationcapacity and the rate of adsorption between DSPA andDSPC are therefore only an effect of the headgroup. Thehighest rate of adsorption of â-lactoglobulin into a phos-pholipidmonolayerwas observed forDSPAat pH7. Thiswas especially pronounced at higher surface pressures,and consequently it was found that the penetrationcapacity was higher for â-lactoglobulin into a DSPAmonolayer than into a DSPC monolayer (see Table 2).How can a protein with a negative net charge interactwith a monolayer of negatively charged phospholipid? Apossible explanation for this might be that a region richon positively charged residues on the protein surface willbe orientated toward thephospholipid. In fact, it hasbeenshown by Ferry and Oncley26 that â-lactoglobulin has ahigh dipole moment, 730 D, as a result of an inhomoge-neous charge distribution.Therateofadsorptionofâ-lactoglobulin intomonolayers

of both phospholipids increased with the salt concentra-tion, but the trend was more pronounced into a DSPAmonolayer. The rates of adsorption were lower for bothphospholipids at pH 4 compared with pH 7, although thedifferenceswerevery small. This is as expected forDSPC,since it is a zwitterion in the investigated pH range. Thepenetration capacities for DSPA at pH 4 were also(24) Graham, D. E.; Philips, M. C. J. Colloid Interface Sci. 1979, 75,

403-439.(25) MacRitchie, F. J. Colloid Interface Sci. 1981, 79, 461-464. (26) Ferry, J. D.; Oncley, J. L. J. Am. Chem. Soc. 1941, 63, 272-278.

Table 2. Surface Pressures above Which Penetration ofâ-Lactoglobulin from a 10 mM Phosphate Buffer into aPhospholipid Monolayer Did Not Occur (T ) 25 °C)

salt conc (mM) DSPA (-) DSPC (+/-) DPPA (-)

0 21 16 17pH 7.0b 50 22 18 18

150 24 21 180 22 19 22

pH 4.0b 50 21 20 20150 21 24 19.5

a In parentheses are the charges at pH 4.0 and 7.0 estimatedfrom the data given by Marsh,37 where +/- denotes zwitterion.b The isoelectric point of â-lactoglobulin is at pH 5.2,1 andconsequently it carriesanegativenet chargeatpH7.0andapositiveone at pH 4.0.

dΓ/dt ) kadsc0 exp(-ΠA0/kT) (1)

Figure 8. Ln(rate) as a function of Π for the incorporation ofâ-lactoglobulin into a DSPA, DSPC, or DPPAmonolayer on 10mM phosphate buffer, pH 7, with a NaCl concentration of 0mM (O), 50 mM (0), or 150 mM (4). The rate is given inmilligrams per meter squared second, and the protein con-centration was 1.15 mg/L.

Interaction between â-Lactoglobulin and Phospholipids Langmuir, Vol. 12, No. 11, 1996 2795

comparable with those at pH 7, but for DSPC they weresomewhathigher. The influence of salt concentrationwasless forDSPAatpH4 thanat pH7. ForDSPCan increaseof the salt concentration at pH 4 resulted in higherpenetration capacities whereas no effect was found forDSPA.These results can be explained in terms of electrostatic

interactions which have been reported to be importantfor protein-lipid interactions.27,28 Screening of the chargesof both the protein and phospholipids by adding saltgenerally results in reduction of the electrostatic interac-tions. The adsorption will therefore be reduced if it isgoverned by electrostatic attractive forces or facilitated iftheprocess ishamperedbyelectrostatic repulsionbetweenthe molecules at the interface. Since we saw an increasein the adsorption rate with increasing salt concentration,the latter process is likely to be the case under theexperimental conditions used. Furthermore, this effectis smaller for the zwitterionic DSPC monolayers. At pH4, where the protein carries a positive net charge (i.e.p.≈ 5.21), a higher rate of adsorption into the negativelycharged DSPA (pK of 3.229) monolayer might be expecteddue to electrostatic attraction. It is therefore remarkable

that the rate of adsorption into DSPA at pH 7, when bothprotein and lipid carry a negative net charge, was almostthe same at low Π or even higher at high Π. However,as discussed above this might be explained by theinhomogeneous charge distribution. The penetrationcapacity into theDSPAmonolayerwas also unaffected bythe pH and the salt concentration.Effect of the Hydrocarbon Chain Length. The

effects of the lipid hydrocarbon chain length on theadsorption of â-lactoglobulin into phosphatidic acid mono-layers have been studied for DSPA and DPPA. Figure 7shows that the adsorption rates at pH 7 were higher intoDSPA then into DPPA monolayers. This was morepronounced at higher surface pressures and was alsoreflected in the lower penetration capacities of â-lacto-globulin intoDPPAcompared toDSPAmonolayers at thispH (Table 2). The dependence on the salt concentrationat pH 7 was the same for both phospholipid monolayers:higher salt concentration resulted in higher adsorptionrates. At pH 4 the rates of adsorption of â-lactoglobulininto a DPPA monolayer were also lower than those foradsorption into a DSPA monolayer. At this pH thedependence on the salt concentration for the adsorptionof â-lactoglobulin into a DSPA monolayer or DPPAmonolayer was comparable. However, it is notable thatat pH4,whenno saltwas added to the subphase, no linearrelation between ln(rate) andΠwas found forDPPA.Thisis probably due to loss of DPPA or DPPA/â-lactoglobulincomplexes into the subphase, as the samebarrier positionwas not obtained upon compression to the high surfacepressure. From these results it can be concluded thatalso the lengths of the hydrocarbon chains, that ishydrophobic interactions, are likely to promote theincorporation of â-lactoglobulin into phosphatidic acidmonolayers.The role of the hydrophobic interactionmight be linked

to changes in conformation during the adsorption processwhich can facilitate the insertion of â-lactoglobulin intophospholipidmonolayers. Brown et al.30 found thatwhenthe R-helix content of â-lactoglobulin was increased,phosphatidylcholinewasable to bind to theprotein,whichwas not possible with the native form of â-lactoglobulin.They suggested that the hydrocarbon chain of the phos-pholipid interacts with the hydrophobic interior of theR-helix while the hydrophilic part of the protein interactswith theheadgroup. However, the circulardichroismdataof â-lactoglobulin bound to a mixed palmitoyloleoylphos-phatidylcholine (POPC) and palmitoyloleoylphosphati-dylglycerol (POPG) monolayer, reported by Cornell,27showed that the conformationof theproteindidnot changesignificantly compared to that in the bulk solution. Thebinding site of nonpolar molecules like alkanes andaromatic compounds has been located in a hydrophobicregion at the surface of the protein.31-33 This binding siteis not involved in the association of â-lactoglobulin, sincethe binding was equal for monomers, dimers, and octam-ers.Structureof theLipidProteinFilm. It is important

to determine if the proteins at the interface exist inseparate domains without lipids, that is if the proteinadsorbs into its ownmonolayer or if it penetrates into thelipid domains. In our experiments we obtained different

(27) Cornell, D. G.; Patterson, D. L. J. Agric. Food Chem. 1989, 37,1455-1459.

(28) Quinn, P. J.; Dawson, M. C. Biochem. J. 1969, 115, 65-75.

(29) Trauble, H.; Eibl, H. Proc. Natl. Acad. Sci. USA 1974, 71, 214-219.

(30) Brown, E. M.; Carroll, R. J.; Pfeffer, P. E.; Sampugna, J. Lipids1983, 18, 111-118.

(31) Mohammadzadeh, A.; Feeney, R. E.; Smith, L. M. Biochim.Biophys. Acta 1969, 194, 246-255.

(32) Wishnia, A.; Pinder, T. W., Jr. Biochemistry 1966, 5, 1534-1542.

(33) Robillard,K.A.;Wishnia,A.Biochemistry1972,11, 3835-3840.

Figure 9. Ln(rate) as a function of Π for the incorporation ofâ-lactoglobulin into a DSPA, DSPC, or DPPAmonolayer on 10mM phosphate buffer, pH 4, with a NaCl concentration of 0mM (O), 50 mM (0), or 150 mM (4). The rate is given inmilligrams per meter squares second, and the protein concen-tration was 1.15 mg/L.

2796 Langmuir, Vol. 12, No. 11, 1996 Bos and Nylander

rates of adsorptionofâ-lactoglobulin intoDSPAandDPPAmonolayers, although the Π-A isotherms were almostidentical. However, with an inhomogeneous film, whereprotein molecules are adsorbed into different proteindomains, the rates of adsorption into the two types ofphospholipidmonolayerswouldprobablybe the same.Thearea increase for adsorption of proteins into its ownmonolayerwas found to bemuch slower than observed forthe penetration into the phospholipid layers (data notshown). Furthermore, we observed penetration of â-lac-toglobulin into the phospholipid monolayer at pressureshigher than the equilibrium surface pressure (15 mN/mafter 2 h) obtained for the adsorption of the protein.Cornell et al.27,34 observed penetration of â-lactoglobulin,R-lactalbumin, or BSA into mixed monolayers of POPCand POPG at such high lipid pressure that they found itunlikely that the proteins could penetrate into a proteinlayer at this pressure. Thus, they concluded that theformation of pure protein patches is unlikely and thatportions of the protein are suggested to be intercalated inthe lipid monolayer. The rates of adsorption of â-lacto-globulin into phospholipidmonolayers were also found tobe independent of the history of the lipid film.Cornell andCarroll35 studied themiscibility inprotein-

lipid monolayers for various proteins, among them â-lac-toglobulin. They found that only lipids with the chainsin the liquid state, such as egg-yolk phosphatidic acid,dioleoylphosphatidylcholine, and dioleoylphosphatidyle-thanolamine, gave homogeneous films with â-lactoglo-bulin, whereas DPPA and DSPC formed heterogeneousfilms. Heckl et al.36 used fluorescencemicroscopy togetherwith the surface film balance technique to study thestructure of mixed films of phospholipids with chains inthe crystalline state and cytochrome c andb. Itwas foundthat proteins were mainly located in the fluid membranephase which coexisted with solid lipid domains withoutprotein. The penetration in the lipid monolayer wasreduced with increasing pressure. Thus, their findingsare ingoodagreementwith theobservations in thepresentstudy, where we also used lipids with crystalline chains.

Comparison with Dispersed Phospholipid Sys-tems. It is interesting to compare our results with ourprevious study of the solution behavior of â-lactoglobulinmixedwith the same pure andwell defined phospholipidsas used in this study.16 We used differential scanningcalorimetry (DSC) to study how and under which condi-tions an interaction between â-lactoglobulin and phos-pholipids would affect the thermal stability of â-lacto-globulin. It was found that the thermally inducedunfolding of â-lactoglobulin was shifted to higher tem-peratures in the presence of DPPA and DSPA. No shiftin denaturation temperature was observed for the otherdistearoyl phospholipids like DSPE, DSPC, or DSPG.Distearoylphosphatidic acid from egg yolk, with unsatur-ated hydrocarbon chains, showed no interaction withâ-lactoglobulin. Our results agree very well with ourearlier study, where it was found that both the hydro-carbon chain as well as the polar headgroup determinethe occurrence of an interaction. However, we did notfind the same strong interaction between a phosphatidicacid with a shorter chain length (DPPA) and â-lactoglo-bulin as reported earlier.16

Conclusions

This study shows that the interaction between â-lac-toglobulin andphospholipids at a liquid interface dependson the headgroup and on the hydrocarbon chain of thephospholipid, suggesting that not only electrostatic butalso hydrophobic interactions are needed for the interac-tion. A conformational or orientational change of theproteinmight benecessary for the protein to interactwiththe lipid, resulting inaprotein-lipid complex. Therefore,an initially electrostatic attraction between polar groupsmight be followed up by hydrophobic interactions causedby conformational or orientational changes of the protein.

Acknowledgment. Prof. Kåre Larsson, Prof. Valde-maras Razumas, Prof. Juan M. Rodriquez Patino, andDr. Anders Carlsson are thanked for their valuablediscussions and comments during this study. The finan-cial support obtained from the European Community(Grant HCM ERBCHRXT930322) via the Swedish Natu-ral Science Research Council and from the SwedishCouncil for Forestry and Agricultural Research isacknowledged.

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(34) Cornell, D. G.; Patterson, D. L.; Hoban, N. J. Colloid InterfaceSci. 1990, 140, 428-435.

(35) Cornell, D. G.; Carroll, R. J. J. Colloid Interface Sci. 1985, 108,226-233.

(36) Heckl, W. M.; Zaba, B. N.; Mohwald, H. Biochim. Biophys. Acta1987, 903, 166-176.

(37) Marsh,D.CRCHandbook ofLipidBilayers; CRCPress: Boston,1990.

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